Charles S. Craik

Global landscape of HIV-human protein complexes

Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host’s cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV–human protein–protein interactions involving 435 individual human proteins, with ∼40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.

Evolutionary Divergence of Substrate Specificity within the Chymotrypsin-like Serine Protease Fold

One of the enduring paradigms in enzymology is the theme of evolutionary divergence in substrate specificity from a parental enzyme possessing a prototypical fold. Within the fold of each divergent enzyme is embedded the catalytic machinery essential to providing rate enhancement for an identical reaction, and the particular amino acids directly involved in this function are expected to be highly conserved and invariant in their spatial positions. Among the remaining residues will be those required for the overall three-dimensional structure. Such residues are found largely within the hydrophobic core of the enzyme and, within a family, may show covariance according to the thermodynamic determinants specifying stability and the informational determinants specifying uniqueness of the fold. The remaining amino acids of the enzymes then constitute much of the raw material for evolutionary adaptation to novel selectivity. Many of these can be expected to be found interacting with distal, variable, specificitydetermining portions of the substrates in modes that are unique to each enzyme of the family. The core structural residues may also contribute to generating new specificity, by virtue of second or third shell interactions with the amino acids directly contacting substrate. Here we describe structure-function relationships in one of the largest and most comprehensively studied of all enzyme families, the serine proteases with a chymotrypsin fold. Over 20 unique structures have been determined to date, and the number of available sequences exceeds 500 (for a comprehensive review, see Ref. 2). All of the enzymes possess an identical fold consisting of two b-barrels, with the catalytic Ser, His, and Asp amino acids found at the interface of the two domains. Common features present in all structures, including five enzyme-substrate hydrogen bonds at positions P1 and P3, serve to properly juxtapose the scissile peptide bond adjacent to the Ser-His catalytic couple, such that the nucleophilic Ser O-g is accurately positioned for attack. Given these conserved interactions in the direct vicinity of the reacting groups, the more distal contacts sharply diverge. We describe the structural themes that embody this divergence together with some of the most important enzymological data necessary to the structure-function correlation. One important theme that emerges is that of catalytic register (3); the divergence in distal interactions in different enzymes must be such as to still permit accurate substrate alignment. Another observation is that many structural determinants controlling specificity reside on surface loops, and this allows for the possibility of rapid and varied evolutionary divergence with conservation of the overall tertiary fold. Divergence of substrate specificity within the context of a common structural framework represents only one mechanism by which nature is able to evolve new activities. Another mechanism involves incorporation of new catalytic groups within an active site, such that the same scaffold can carry out a range of chemistries with just a single step common to different members of the family. This theme is elaborated in the review by Babbitt and Gerlt (4). It is also possible to obtain information on the requirements for a common catalytic function by studying examples of convergent evolution, where the same chemical reaction is carried out by very different scaffolds. Serine proteases represent a paradigm in this respect as well; besides the chymotrypsin-fold enzymes, there are now four other known natural folds that possess the requisite catalytic determinants in similar spatial positions (5–10). Study of these structures and redesigned versions of trypsin (11) shows that the catalytic Asp can adopt virtually any position with respect to the Ser-His dyad, suggesting that the classical “catalytic triad” of Ser, His, and Asp residues (Fig. 1) can in fact be better described as the juxtaposition of two dyads: Ser-His and His-Asp (6).

Cathepsin G Activates Protease-activated Receptor-4 in Human Platelets

Of the four known protease-activated receptors (PARs), PAR1 and PAR4 are expressed by human platelets and mediate thrombin signaling. Whether these receptors are redundant, interact, or play at least partially distinct roles is unknown. It is possible that PAR1 and/or PAR4 might confer responsiveness to proteases other than thrombin. The neutrophil granule protease, cathepsin G, is known to cause platelet secretion and aggregation. We now report that this action of cathepsin G is mediated by PAR4. Cathepsin G triggered calcium mobilization in PAR4-transfected fibroblasts, PAR4-expressingXenopus oocytes, and washed human platelets. An antibody raised against the PAR4 thrombin cleavage site blocked platelet activation by cathepsin G but not other agonists. Desensitization with a PAR4 activating peptide had a similar effect. By contrast, inhibition of PAR1 function had no effect on platelet responses to cathepsin G. When neutrophils were present, the neutrophil agonist fMet-Leu-Phe triggered calcium signaling in Fura-2-loaded platelets. Strikingly, this neutrophil-dependent platelet activation was blocked by the PAR4 antibody. These data show that PAR4 mediates platelet responses to cathepsin G and support the hypothesis that cathepsin G might mediate neutrophil-platelet interactions at sites of vascular injury or inflammation.

Substrate Profiling of Cysteine Proteases Using a Combinatorial Peptide Library Identifies Functionally Unique Specificities

The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.

Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin

We have developed a strategy for the synthesis of positional-scanning synthetic combinatorial libraries (PS-SCL) that does not depend on the identity of the P1 substituent. To demonstrate the strategy, we synthesized a tetrapeptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized. The 6,859 members of the library were synthesized on solid support with an alkane sulfonamide linker, and then displaced from the solid support by condensation with a fluorogenic 7-amino-4-methylcoumarin-derivatized lysine. This library was used to determine the extended substrate specificities of two trypsin-like enzymes, plasmin and thrombin, which are involved in the blood coagulation pathway. The optimal P4 to P2 substrate specificity for plasmin was P4-Lys/Nle (norleucine)/Val/Ile/Phe, P3-Xaa, and P2-Tyr/Phe/Trp. This cleavage sequence has recently been identified in some of plasmins physiological substrates. The optimal P4 to P2 extended substrate sequence determined for thrombin was P4-Nle/Leu/Ile/Phe/Val, P3-Xaa, and P2-Pro, a sequence found in many of the physiological substrates of thrombin. Single-substrate kinetic analysis of plasmin and thrombin was used to validate the substrate preferences resulting from the PS-SCL. By three-dimensional structural modeling of the substrates into the active sites of plasmin and thrombin, we identified potential determinants of the defined substrate specificity. This method is amenable to the incorporation of diverse substituents at the P1 position for exploring molecular recognition elements in proteolytic enzymes.

Vif hijacks CBF-β to degrade APOBEC3G and promote HIV-1 infection

Restriction factors, such as the retroviral complementary DNA deaminase APOBEC3G, are cellular proteins that dominantly block virus replication. The AIDS virus, human immunodeficiency virus type 1 (HIV-1), produces the accessory factor Vif, which counteracts the host’s antiviral defence by hijacking a ubiquitin ligase complex, containing CUL5, ELOC, ELOB and a RING-box protein, and targeting APOBEC3G for degradation. Here we reveal, using an affinity tag/purification mass spectrometry approach, that Vif additionally recruits the transcription cofactor CBF-β to this ubiquitin ligase complex. CBF-β, which normally functions in concert with RUNX DNA binding proteins, allows the reconstitution of a recombinant six-protein assembly that elicits specific polyubiquitination activity with APOBEC3G, but not the related deaminase APOBEC3A. Using RNA knockdown and genetic complementation studies, we also demonstrate that CBF-β is required for Vif-mediated degradation of APOBEC3G and therefore for preserving HIV-1 infectivity. Finally, simian immunodeficiency virus (SIV) Vif also binds to and requires CBF-β to degrade rhesus macaque APOBEC3G, indicating functional conservation. Methods of disrupting the CBF-β–Vif interaction might enable HIV-1 restriction and provide a supplement to current antiviral therapies that primarily target viral proteins.

Definition and Redesign of the Extended Substrate Specificity of Granzyme B

Granzyme B is a protease involved in the induction of rapid target cell death by cytotoxic lymphocytes. Definition of the substrate specificity of granzyme B allows for the identification of in vivo substrates in this process. By using the combinatorial methods of synthetic substrate libraries and substrate-phage display, an optimal substrate for granzyme B that spans over six subsites was determined to be Ile-Glu-Xaa-(Asp↓Xaa)-Gly, with cleavage of the Asp↓Xaa peptide bond. Granzyme B proteolysis was shown to be highly dependent on the length and sequence of the substrate, supporting the role of granzyme B as a regulatory protease. Arginine 192 was identified as a determinant of P3-Glu and P1-Asp substrate specificity. Mutagenesis of arginine 192 to glutamate reversed the preference for negatively charged amino acids at P3 to positively charged amino acids. The preferred substrate sequence matches the activation sites of caspase 3 and caspase 7 and thus is consistent with the role of granzyme B in activation of these proteases during apoptosis. The caspase substrate poly(ADP)-ribose polymerase is cleaved by granzyme B in a cell-free assay at two sites that resemble the granzyme B specificity determined by the combinatorial methods. Many caspase substrates contain granzyme B cleavage sites and are proposed as potential granzyme B targets, suggesting a redundant function with certain caspases.

Molecular cloning and primary structure of human 15-lipoxygenase

A full-length cDNA encoding 15-lipoxygenase has been isolated from a human reticulocyte cDNA library. The predicted primary structure of the enzyme exhibits a sequence similarity of 61% and 45% with human 5-lipoxygenase and the soybean lipoxygenase isoenzyme I, respectively. When all three lipoxygenases are aligned, there are two distinct regions of significant sequence identity including a cluster of five histidine residues conserved in all three lipoxygenases. Because histidines can serve as ligands for the enzymatically active iron, this region may be critical to enzymatic function. These results provide a basis for exploring functional domains of lipoxygenases.

Inhibition of the HIV-1 and HIV-2 proteases by curcumin and curcumin boron complexes

Curcumin, a relatively non-toxic natural product isolated from Curcuma longa, is a modest inhibitor of the HIV-1 (IC50 = 100 microM) and HIV-2 (IC50 = 250 microM) proteases. Simple modifications of the curcumin structure raise the IC50 value but complexes of the central dihydroxy groups of curcumin with boron lower the IC50 to a value as low as 6 microM. The boron complexes are also time-dependent inactivators of the HIV proteases. The increased affinity of the boron complexes may reflect binding of the orthogonal domains of the inhibitor in interesecting sites within the substrate-binding cavity of the enzyme, while activation of the alpha, beta-unsaturated carbonyl group of curcumin by chelation to boron probably accounts for time-dependent inhibition of the enzyme.

Catalytic properties and inhibition of proline-specific dipeptidyl peptidases II, IV and VII

There is currently intense interest in the emerging group of proline-specific dipeptidases, and their roles in the regulation of biological processes. Dipeptidyl peptidase IV (DPP-IV) is involved in glucose metabolism by contributing to the regulation of glucagon family peptides and has emerged as a potential target for the treatment of metabolic diseases. Two other proline-specific dipeptidases, DPP-VII (also known as quiescent cell proline dipeptidase) and DPP-II, have unknown functions and have recently been suggested to be identical proteases based on a sequence comparison of human DPP-VII and rat DPP-II (78% identity) [Araki, Li, Yamamoto, Haneda, Nishi, Kikkawa and Ohkubo (2001) J. Biochem. 129, 279-288; Fukasawa, Fukasawa, Higaki, Shiina, Ohno, Ito, Otogoto and Ota (2001) Biochem. J. 353, 283-290]. To facilitate the identification of selective substrates and inhibitors for these enzymes, a complete biochemical profile of these enzymes was obtained. The pH profiles, substrate specificities as determined by positional scanning, Michaelis-Menten constants and inhibition profiles for DPP-VII and DPP-II were shown to be virtually identical, strongly supporting the hypothesis that they are the same protease. In addition, substrate specificities, catalytic constants and IC(50) values were shown to be markedly different from those of DPP-IV. Selective DPP-IV and DPP-VII substrates were identified and they can be used to design selective inhibitors and probe further into the biology of these enzymes.